Several reports have appeared recently in the literature detailing cases in which gastrointestinal (GI) disease, often with significant associated morbidity, occurred in AIDS patients infected with microsporidial parasites. As a consequence of these reports, there has been an increased awareness among clinicians of the potential importance of microsporidia as GI pathogens, and attention is now being focused on methods for both diagnosis and treatment. There is considerable interest in determining the diagnostic value of stool specimens for detecting GI disease due to microsporidia, and a variety of staining modalities have been evaluated for their ability to visualize microsporidia in stool preparations. Unfortunately, the various species of microsporidia that infect humans can be identified accurately only by visualizing characteristic subcellular morphologic features of organisms in electron micrographs of tissue specimens. Since the efficacy of treatment regimens currently in development may be somewhat species-specific, and because invasive procedures are necessary to obtain appropriate specimens for electron microscopy, a more widely available approach to identification is clearly required. We have published a report describing our polymerase chain reaction (PCR) assay for amplification of microsporidial rRNA genes followed by restriction endonuclease digestion of PCR products as a method for identifying microsporidia in fresh stool. The PCR primers used in the assay will amplify all of the currently identified microsporidial human pathogens. The assay is undergoing modification to allow detection of microsporidia in formalin-fixed stool specimens. A dot-blot format using PCR-amplified DNA and species-specific DNA probes has been developed for use in microsporidial speciation. The final stage of the project will be to determine the sensitivity of our assay and the length of time specimens can be stored in formalin and still allow detection of the microsporidia.